Analogues of phosphatidylcholine or phosphatidyl ethanol amines

ABSTRACT

A compound of formula: ##STR1## in which X 1  is a reactive group that can react to form a covalent bond with a reactive group on the surface of a material to be rendered biocompatible, X 2  is a group -0.sup.⊖ or a precursor of such a group, n is 2, 3 or 4, Y is a group --N.sup.⊕ R 3  A.sup.⊖ wherein each R, which may be the same or different, is a C 1  -C 4  alkyl group and A.sup.⊖ is an anion present when X 2  is an electrically neutral group or Y is ##STR2## wherein R 1  together with X 2  forms a direct bond between the nitrogen and the phosphorus atoms.

This is a continuation of application Ser. No. 07/114,762, filed Oct.29, 1987, now abandoned.

Which is a division of application Ser. No. 778,185, filed Sept. 18,1985.

This invention relates to biocompatible surfaces and to new compoundsuseful in the production of such surfaces.

The clinical use of blood contacting devices and prostheses is of majorimportance today in cardiovascular surgery and other fields of medicine.Heart valves and blood vessel prostheses, balloon pumps and cathetersare being implanted in daily surgical practice to restore or diagnosecardiovascular function. Artificial organs are routinely employed inblood detoxification by absorptive haemoperfusion and in oxygenation(membrane oxygenators and heart-lung devices). Considerable effort andcapital is invested in Europe and the U.S.A. in the development andexperimental evolution of an implantable artificial heart system. Thedevices are generally constructed from polymeric materials and when inuse, a blood-polymer contact is present. This contact will cause areaction in the recirculating blood, which, depending on the choice ofmaterial, the design parameter, the flow or the addition of theanticoagulants, may lead to protein deposition, adhesion and destructionof red blood cells (haemolysis), platelet (thrombocyte) adhesion andaggregation and blood coagulation leading to the formation of ahaemostatic plug (thrombus). The occurrence of thromboembolism incardiovascular surgery continues to be a problem, notwithstandingroutine treatment with anticoagulants. For these reasons the search forbiocompatible non-thrombogenic materials has been an important researchobjective over the last two decades.

Our concept is to try to mimic, as far as possible, the interfacialcharacteristics of the outer cell surface of red blood cells andplatelets. The simplest common factor of all these surfaces is the lipidof the biological membrane.

Biological membranes are important in all areas of the body. Every cellhas an outer membrane and within the cell there are membranes that actto compartmentalise the various organelles, e.g., the mitochondria,nucleus and endoplasmic reticulum. Membranes are particularly importantfeatures of the blood cells, e.g. erthrocytes and leucocytes. Thevarious cell membranes, including those of red blood cells, are allbuilt upon an asymmetric lipid matrix of polar lipids in which theintrinsic proteins are distributed. The outer surface of the lipidmatrix consists of both phosphatidyl choline lipids and sphingomyelinlipids. Both of these classes of lipid have the same polar group:##STR3##

This polar surface is a common feature of the outer surface of red bloodcells, platelets, lymphocytes, etc. The inner surface is different andusually contains a predominance of the negatively charged lipids.

In recent years there has been considerable work on the physicalchemistry of phospholipids and membranes. (Chapman, D., Q. Rev.Biophys., 8, 185, 1975). Studies of cell systems have shown (Zwaal etal., Nature, 268, 358-360, 1977) that pro-coagulant lipids occur on theinner cell surface but not on the outer surfaces.

Our new approach to this problem is therefore to mimic the lipid polarportion of the cell membrane's outer surface by altering surfacecharacteristics of existing materials, e.g. glasses and polymers bychemical modifications.

The aim is chemically to modify some existing materials so that covalentlinkages are formed containing these polar groups. This retains themechanical properties of the material whilst the interfacial propertiesare changed to mimic those of membrane surfaces.

In some of our recent studies (Hayward & Chapman Biomaterials, 5,135-141, 1984) we have examined surface coatings containing theappropriate polar groups. The haemocompatibility of liposomalpreparations was estimated by comparing the recalcification clottingtimes of citrated pooled normal plasma in the presence of assorted lipiddispersions. A brain lipid extract (containing large amounts ofnegatively charged phospholipids) markedly accelerated the rate of clotformation in a concentration-dependent manner. In contrast, liposomesprepared from dimyristoyl phosphatidylcholine did not reduce the blankclotting times. Similarly, clot formation was not affected bydiacetylenic phosphatidylcholine when present in either monomeric orpolymeric form. These results support our concept for biocompatiblesurfaces. It is clear however that covalent linkages of the polar groupsto the treated material are necessary for maximum mechanical stability.As far as we are aware, there has been no modification of the type thatwe are proposing.

We have discovered a group of new compounds which are derivatives oranalogues of phosphatidylcholine or phosphatidyl ethanolamine that canbe covalently linked to the surface which is to be renderedbiocompatible so as to deposit a phosphatidylcholine- or phosphatidylethanolamine-type of residue on the surface. New compounds of thepresent invention are those of the formula: ##STR4## in which X¹ is areactive group that can react to form a covalent bond with a reactivegroup on the surface of a material to be rendered biocompatible, X² is agroup -O.sup.⊖ or a precursor of such a group, n is 2, 3 or 4, Y is agroup --N.sup.⊕ R₃ A.sup.⊖ wherein each R, which may be the same ordifferent, is a C₁ -C₄ alkyl group and A.sup.⊖ is an anion present whenX² is an electrically neutral group or Y is ##STR5## wherein R¹ togetherwith X² forms a direct bond between the nitrogen and the phosphorousatoms.

In the compounds of the invention, it is preferred that each Rrepresents methyl and that n is 2 so that a phosphatidylcholine residueis introduced onto the surface but analogues and homologues ofphosphatidylcholine of the type defined can equally well be introduced.Alternatively, or preferably in combination therewith, a phosphatidylethanolamine-type residue is introduced onto the surface.

The exact chemical nature of the group X¹ will depend upon the chemicalnature of the reactive group on the surface to be renderedbiocompatible. Almost all surfaces that one might wish to renderbiocompatible normally contain free reactive alcoholic hydroxy groups ontheir surface or are surfaces onto which such free alcoholic hydroxygroups can be readily introduced, e.g using an alkali metal hydroxide tohydrolyse a surface ester group or halogeno group. Consequently, X¹ willnormally be a group that will react with an alcoholic hydroxy group toform a covalent link, typically by forming a phosphonic acid ester groupfrom the alcoholic hydroxy group and the phosphonic acid residue of thephosphatidylcholine- or phosphatidyl ethanolamine-type material. Suchphosphonic ester groups can be prepared from compounds of the inventionin which X¹ represents halogen, particularly chlorine although,depending upon the reactivity of the alcoholic hydroxy group, otherhalogens such as fluorine or bromine can also be used.

As an alternative to the use of halogeno derivatives X¹ may alsorepresent a group of the formula: ##STR6## each R, which may be the sameor different, represents a C₁ -C₄ alkyl group, preferably both methyl,and X³ represents a group that will react with the reactive group on thesurface to be rendered biocompatible with the formation of a covalentlinkage. As in the case of X¹, the exact chemical nature of X³ willdepend upon the nature of the reactive group on the surface to berendered biocompatible but, for the reasons mentioned above, thereactive group on the surface to be rendered biocompatible will normallybe an alcoholic hydroxy group, and, again as mentioned above, thisindicates that the group X³ will conveniently be a halogeno group,typically chlorine although again, depending upon the reactivity of thealcoholic hydroxy group, other halogens such as fluorine or bromine canbe used.

In a further embodiment of the invention, X¹ can represent a group ofthe formulae: ##STR7## where y is an integer of 1 to 10 and R and X³ areas defined above.

The group X² will normally be -O.sup.⊖ so that the modified surfacecarries a phosphatidylcholine- or phosphatidyl ethanolamine-type residuebut it is often convenient to have the group X² in the new compounds ofthe invention representing a precursor of the -O.sup.⊖ group, e.g.halogen such as chlorine so that this precursor group is converted to-O.sup.⊖ before, during or after formation of the covalent linkage bythe reaction of the group X¹.

Anion A.sup.⊖ when required to be associated with the new compounds ofthe invention can be any anion, preferably a biocompatible anion. Thisanion can be the anion of an inorganic or organic acid and is typicallya halide anion such as chloride or alternatively the anion of analkanoic acid such as acetic acid.

The new compounds of the invention can be prepared by reacting acompound of the formula:

    Y(CH.sub.2).sub.n -OH                                      VI

with a phosphorus oxyhalide e.g. POCl₃ to give compounds of the formulaI in which X¹ represents halogen and, when Y is a group -N.sup.⊕R₃.A.sup.⊖, X² is also halogen. Analogues of phosphorus oxyhalides maybe used in order to produce derivatives of formula I in which X¹ and X²represent other reactive groups or alternatively, reaction can first becarried out with phosphorus oxyhalide to give a dihalophosphate and oneor both of the halogeno residues can subsequently be converted bymethods known per se into other reactive groups so that X¹ can reactwith the reactive group on the surface to be rendered biocompatible withthe formation of the covalent link.

Compounds of the invention where X¹ represents a group of the formula IIas indicated above can be obtained by reaction of a compound of formulaVI wherein Y is a group --NR₃.A with e.g. phosphorus oxychloride asindicated above to form a dichlorophosphate of acetylcholine or ananalogue thereof. Hydrolysis of the chlorine residues, e.g. by treatmentwith aqueous sodium bicarbonate or sodium carbonate converts thedichlorophosphate into a derivative of phosphatidylcholine or ananalogue thereof of the formula: ##STR8## and this may be reacted with asilane of formula: ##STR9## where R and X³ are as defined above and halis a halogeno group to introduce the group II as defined above.

Compounds of the invention wherein Y is a group --N.sup.⊕ R₃.A.sup.⊖ andX¹ is a group of formula III and V can be prepared by reacting acompound of the formula: ##STR10## with

    T10R                                                       IIIA

or IIA respectively.

Compound VIII is prepared by reacting I with an ethyl vinyl ether monoprotected diols of formula: ##STR11## followed by removal of theprotective group with acidified ice water, concentration and sodiumbicarbonate treatment.

Compounds wherein Y is a group --NR₃.A and X¹ is a group of formula IVare prepared by reacting I with an alkanolamine followed by the cleavageof the oxazaphospholane X thus formed using aqueous acid followed by abase such as sodium bicarbonate: ##STR12## Compound of formula (I)wherein Y is ##STR13## may be produced in the same manner usingappropriate starting materials thus, for instance phosphorus oxychloridereacts with ethanolamine to form 2-chloro-2-oxo-1,2,3-oxazapholane asfollows: ##STR14##

All the compounds of the invention will react with hydroxy groups in thesurface to be rendered biocompatible to bind the phosphatidylcholine- orphosphatidyl ethanolamine-type covalently to the surface exceptcompounds of the invention where X¹ is a group of formula III or V whichwill react with surface halogens and acid chloride groups respectively.In all cases the phosphatidylcholine- or phosphatidyl ethanolamine-typegroup becomes covalently linked to the surface.

In the case of 2-chloro-2-oxo-1,2,3-oxazapholane which contains areactive chlorine atom, when brought in contact with an hydroxyl group,this is deposited as follows: ##STR15##

The oxazapholane ring is finally cleaved with dilute acetic acid toobtain the ethanolamine residue: ##STR16## Similar processes may beemployed for introducing other phosphatidyl ethanolamine-type groupsonto a surface.

In accordance with a further feature of the invention, there is provideda process for rendering a surface biocompatible which comprises applyingto the surface a compound of formula I under conditions such that thegroup X¹ reacts to form a covalent linkage with a reactive group on thesurface to be rendered biocompatible.

The compounds of the invention are not very soluble in solvents such aschlorofom, petroleum ether, carbon tetrachloride or tetrahydrofuran andtend to react with solvents in which they are appreciably soluble, e.g.water or alkanols, so that it is preferred to use the compounds inundiluted state for the treatment of the surfaces. Although compounds inwhich X¹ is a group IV do not react with either water or alkanols, thesurface acid chloride group (which is meant to react with X¹ in thesecases) will react with these solvents.

The present invention is applicable in principle to the treatment of thesurface of any prosthesis to be introduced into the human or animal bodyor any surface which is to be brought into contact with body fluids,e.g. blood on an extra-corporeal basis, cells or tissues in culture. Itmay also be advantageous to treat surfaces of equipment, especiallyculture vessels, used in cell and tissue culture, in order to providebiocompatible surfaces. In order to demonstrate the benefit of thepresent invention, we have carried out experiments with glass, polyvinylalcohol, perspex, polyhema, cellulose acetate and PTFE surfaces and withsurfaces of polymethylmethacrylate which have been surface modified toensure the presence of alcoholic hydroxy groups in the surface. Howeversurfaces of other materials such as metals, or plastics which arepolymers or copolymers based on various polyacrylic or polyvinylmaterials and others containing olefinic bonds are also suitable formodification, if necessary after replacing the double bonds by, forexample, halogeno, hydroxy or acid chloride groups, in accordance withthe present invention.

The following Examples are given to illustrate the invention.

EXAMPLE 1 Preparation of choline acetate dichlorophosphate (I)(a):##STR17##

Phosphorus oxychloride (23 g; 0.15 mole) was dissolved in anhydrouscarbon tetrachloride (50 ml), placed in a bath containing ice/saltmixture and was treated with a dropwise addition of acetylcholine (16.3g; 0.1 mole) over 30 minutes. During this addition, a slow stream ofdried nitrogen was passed through the mixture, which was stirredthoroughly throughout the reaction time. After the addition ofacetylcholine, the mixture was stirred for an additional hour at 0° C.with continuous nitrogen message.

The upper layer of the reaction mixture was collected, washed once withanhydrous carbon tetrachloride (50 ml) and was then freed from thevolatile matter (40° C., 15 mm Hg) to obtain the yellow oil (I)(a) inmore than 70% yield. The substance was highly reactive towards waterwith the evolution of hydrogen chloride and gradually decomposed onstanding but can be stored for up to 3 weeks under dry nitrogen in anair-tight glass container.

EXAMPLE 2 Alternative Preparation of Compound (1)(b)

Acetylcholine dichlorophosphate (Ia) (10 g), obtained as described inExample 1, was placed in an ice bath, stirred and then treated withdrops of ice water (10-15 ml). Most of the unreacted water wasevaporated (60° C.; 15 mm Hg) the residue dissolved in methanol (40 ml)and treated with solid sodium bicarbonate until no more effervescencewas observed. The solid was removed by filtration, washed once withmethanol (10 ml) and the solid discarded. The combined filtrate wasevaporated to dryness (60° C.; 15 mm Hg) and the residue, which was freefrom volatile matter, was stored overnight in a vacuum desiccator overphosphorus pentoxide at 0.1 mm.

Phosphatidylcholine, obtained as above, was added dropwise to avigorously stirred dichlorodimethyl silane (50 ml) over 45 minutes. TheHCl gas produced was flushed out by passing a stream of dried nitrogenthrough the reaction mixture. After the addition of phosphatidylcholinewas completed, the mixture was kept stirring for an additional 30minutes. Volatile matter of the reaction mixture was distilled off (50°C.; 15 mm Hg) which was mostly the unreacted silane with some HCl gas,to obtain (I)(b) as greyish red, viscous, non-volatile matter. It wasfound to be active towards water.

EXAMPLE 3 Preparation of ethylene glycol phosphatidylcholine (I)(c) (i)Half protected ethyl vinyl adduct of ethylene glycol(1-Ethoxyethyl-2-hydroxy ethyl ether) ##STR18##

Dried ethylene glycol (124 g; 2 moles) was placed in ice/salt mixture,stirred and treated with a dropwise addition of ethyl vinyl ether (72 g;1.0 mole) in the presence of p-toluene sulphonic acid (0.25 g) over twohours. The mixture was then stirred for an additional hour at 0° C. Theproduct distilled at 80°-86° C.

(ii) Ethylene glycol phosphatidylcholine (I)(c)

Compound (I)(a) (28.1 g; 0.1 mole) was stirred thoroughly and treatedwith a dropwise addition of 1-ethoxyethyl-2-hydroxyethylether (13.4 g;0.1 mole) over one hour. External cooling was applied in order tocontrol the temperature at around 15° C. The evolved hydrochloric acidwas removed from the reaction mixture by a constant stream of driednitrogen. When the addition was completed, the mixture was stirred foranother 15 minutes at room temperature.

Concentrated hydrochloric acid (2 ml) and crushed ice (100 g) mixturewas added and stirring continued. After about 15 minutes stirring themixture appeared to have increased its temperature as well as becomedarker in colour. (Larger scale, e.g. 1 mole, reactions become hot atthis stage but the temperature must be controlled to around roomtemperature).

Volatile materials were evaporated (60°-70° C.; 15 mm Hg) and theresidue was dissolved in methanol (75 ml). The solution was treated withsolid sodium bicarbonate until no more effervescence was noticed. Thesolid was filtered and was wahsed with methanol (10 ml). The combinedfiltrate was evaporated to dryness (70° C.; 15 mm) to free it from allthe volatile matter. It was then stored over P₂ O₅ at 0.1 mm Hg for 24hours.

EXAMPLE 4 Preparation of Phosphatidylcholine ethylene glycol thallate(I)(d) ##STR19##

Ethylene glycol phosphatidylcholine (I)(c) (5.67 g; 0.025 mole) wasdissolved in anhydrous absolute alcohol (25 ml), stirred and was treatedwith thallous ethoxide (6.22 g; 0.025 mole). A white instantaneousprecipitate was formed which was found to be very sparingly soluble inthe solvent used. It was also found to be insoluble in acetronitrite andbenzene. The substance decomposed and dissolved in water.

EXAMPLE 5 Preparation of phosphatidylcholine ethylene glycol dimethylsilyl chloride (I)(e): ##STR20##

Ethylene glycol phosphatidylcholine (I)(c) (22.7 g; 0.1 mole) was addeddropwise into thoroughly stirred dichlorodimethylsilane (129 g; 1.0mole). External cooling was applied to keep the temperature at about 20°C. The addition was completed in 30 minutes and the stirring wascontinued for another 30 minutes after which the mixture was freed fromall volatile components (40° C.; 15 mm Hg) to obtain (I)(e) as dirtygreyish gelatinous viscous liquid. The substance reacted with water withthe evolution of hydrochloric acid.

EXAMPLE 6 Preparation of phosphatidylcholine ethanolamine (I)(f):##STR21##

Choline acetate dichlorophosphate (28.1 g; 0.1 mole) was thoroughlystirred, cooled in an ice/salt bath and was treated very carefully withsmall drops of ethanolamine (61 g; 0.1 mole) over one hour. During thisoperation a slow stream of dried nitrogen was passed through thereaction vessel to remove the dense cloud formed. The mixture, after 10minutes, settled into a reddish semisolid mass. It was then treated withdilute acetic acid (10% w/v, 100 ml) in which it failed to dissolve. Ahomogeneous solution, was however, obtained when it was allowed to reactovernight. Volatile substances were evaporated (50°-60° C.; 15 mm Hg)and the residue was dissolved in methanol. It was then treated withsolid sodium bicarbonate and the solid removed by filtration. The liquidwas again freed from the volatile matter and the residue thus obtained(I)(f) was stored for 24 hours at 0.1 mm Hg, over P₂ O₅.

EXAMPLE 7

Reaction of (I)(a) with

(i) glass

(ii) polyvinyl alcohol

(iii) polyhema

and

(iv) cellulose acetate.

All of the above mentioned substances have free --OH groups on thesurface.

A thin film of compound (I)(a) was applied to the materials named above.They were placed in a desiccator over P₂ O₅ for up to 5 minutes afterwhich the excess of the reagent was washed off with water followed byethanol and sodium bicarbonate solution (5% w/v). They were washed withwater again followed by alcohol and dried with warm air.

The procedure can be shown as an equation as follows: ##STR22##

Treated polyvinyl alcohol, polyhema and celluslose acetate sheet gavepositive ESCA analysis for phosphorus and nitrogen. The glass beads gavepositive phosphate determinations by using untreated beads as reference.

EXAMPLE 8 Reaction of (I)(b) with (i) glass and (ii) cellulose acetate

The procedures followed for this treatment are given in Example 7.

Cellulose acetate and glass beads gave positive ESCA for phosphorus andnitrogen.

EXAMPLE 9 Reaction of (I)(e) with glass

The procedure followed for this treatment has already been given inExample 7.

Substance (I)(a) is superior to substances (I)(b) and (I)(e) whenorganic materials are required to be treated. The stability of thechemical bond obtained when (I)(a) is reacted with C--OH group issuperior in stability and resembles those which occur naturally (forexample in glycerophosphatidylcholine (GPC)). But the type of linkage(C--O--Si) obtained when (I)(b) and (I)(e) are treated with an organicOH group is less stable to hydrolysis. On the other hand compounds(I)(b) and (I)(e) are superior to (I)(a) when treatment of glass isrequired. The bond (Si--O--P) is susceptible to hydrolysis when (Ia) isused to treat glass surfaces.

EXAMPLE 10 Treatment of perspex with (I)(f)

The procedure can be shown as an equation as follows: ##STR23##

Perspex surface was hydrolysed by stirring perspex pieces in 40% w/vaqueous KOH at 80° C. for 12 hours. Neutralisation of the above treatedperspex with dilute hydrochloric acid generated free --COOH groups onthe surface. These pieces were then placed in thionyl chloride for up to5 seconds and while still wet with thionyl chloride, were dropped in aflask containing (I)(f). The contents of the flask were mixed thoroughlyand the plastic pieces were recovered, washed with hot methanol, waterand methanol again and dried in warm air.

EXAMPLE 11 Treatment of halogenated surfaces with (I)(d):

The procedure can be shown as an equation as follows: ##STR24##

Clean polyvinyl chloride or PTFE pieces were added in a flask containing(I)(d) (produced from 6.22 g of thallous ethoxide) suspended in ethanol(50 ml) and acetonitrile (50 ml). The mixture was stirred and heated(30°-40° C.) under dried nitrogen for 48 hours (PTFE samples were heatedfor 96 hours). The plastic pieces were recovered and washed withdistilled water and alcohol and dried.

EXAMPLE 12 Preparation of 2-chloro-2-oxo-1,2,3-oxazapholane

Phosphorus oxychloride (60 g; 0.39 mole) was diluted with anhydroustetrahydrofuran (100 ml) cooled (0° C.) and treated with a dropwiseaddition of triethylamine (25 g) with vigorous stirring over 30 minutes.A gentle stream of dried nitrogen was passed through the stirredmixture, and with the temperature still maintained at 0° C. the mixturewas treated with small drops of ethanolamine (23.92 g; 0.39 mole) over30 minutes and then stirred for an additional one hour. Volatile matterwas distilled (40° C.; 15 mm Hg) and the gumlike dark orange semisolidwas extracted with dried nitromethane (4×40 ml). The nitromethaneextract was concentrated free from the solvent (50° C.; 15 mm Hg) toobtain the required substance as dark red oil which was highly reactivetowards water.

It was possible to use the freshly dissolved substance in nitromethanesolution for coating the hydroxyl group containing surfaces (such asglass) but the compound became inactive when left for over 24 hours innitromethane solution.

We claim:
 1. A compound of formula ##STR25## in which X¹ is halogen, X²is group --O.sup.⊖ or a precursor of such a group, n is 2, 3 or 4, Y isa group --N⁺ R₃ A⁻ wherein each R which may be the same or different, isa C₁ -C₄ alkyl group and A⁻ is present when X² is an electricallyneutral group.
 2. A compound according to claim 1 wherein X¹ ischlorine.
 3. A compound according to claim 1 wherein Y is --N.sup.⊕ R₃A.sup.⊖ and each R is methyl.
 4. A compound according to claim 3 whereinn is
 2. 5. Choline acetate dichlorophosphate.